A number of diseases are associated with the misfolding and aggregation of proteins and peptides into structures known as amyloid fibrils. This group of misfolding diseases includes for example the neurodegenerative Alzheimer's, Parkinson's and Creutzfeldt-Jacob's diseases (Benson, M. D. et al. Kidney Int. 2008, 74, 218-222). Amyloid fibrils are highly stable, β-sheet structures with similar morphology regardless of what protein they are formed from.
Alzheimer's disease (AD) is the most common cause of dementia, accounting for about 60% of all cases (Fratiglioni, L. et al. Neurology 2000, 54, S10-15). It is a progressive neurodegenerative disorder for which there is no cure. One of the hallmarks of AD is cerebral extracellular deposits, called plaques. These plaques are mainly composed of amyloid fibrils formed from the amyloid β-peptide (Aβ). Aβ is an amphipathic peptide of mainly 40- or 42-residues produced by enzymatic cleavages from an integral membrane protein, the amyloid β precursor protein (AβPP) (Mattson, M. P. Physiol. Rev. 1997, 77, 1081-1132), a protein with no known function. The 40-42 residues long Aβ is invariably present in amyloid plaques found in association with Alzheimer's disease, and formation of Aβ fibrils, through aggregation of peptides in β-strand conformation, is thought to be a major part of the cause of this devastating disease (Goedert, M.; Spillantini, M. G. Science 2006, 314, 777-781). When generated from AβPP, initially Aβ harbours α-helices which are strongly predicted to form β-strands (discordant helices) (Kallberg, Y. et al. J. Biol. Chem. 2001, 276, 12945-12950). It is not settled what intermediate(s) in the pathway leading to fibril formation is the dominant toxic species (Dahlgren, K. N. et al. J Biol Chem 2002, 277, 32046-32053). Evidence is accumulating that prefibrillar soluble aggregates, including species referred to as protofibrils, are more toxic than the mature fibrils (Walsh, D. M. et al. Nature 2002, 416, 535-539).
The structure of amyloid fibrils was recently established (Petkova, A. T. et al. Proc. Natl. Acad. Sci. U.S.A 2002, 99, 16742-16747) and support the indication that a region around positions 17-20 is essential for Aβ fibril formation (Janek, K. et al. Biochemistry 2001, 40, 5457-5463). Obstruction of fibril and/or oligomer/protofibril formation could prevent the occurrence or progression of Alzheimer's disease and several ways are being explored to accomplish this (Roberson, E. D.; Mucke, L. Science 2006, 314, 781-784). Major attempts to prevent fibril toxicity involve active or passive immunisation. These attempts have given promising results in animal models, but also given serious side-effects in clinical trials. An alternative approach involves targeting Aβ fibril formation with low molecular weight compounds. Compounds which can abrogate fibril formation by interfering with peptide-peptide contacts in fibrils have been identified (Soto, C. et al. Nat Med 1998, 4, 822-826). A potential drawback with such compounds is that they not only reduce fibril formation, but may also increase the amounts of oligomers/protofibrils (that could be toxic).
In aqueous solution, Aβ is found to be mainly disordered but shows non-random conformations in some regions. Hydrophobic interactions have been indicated between side chains of residues 16-24 and a turn-like structure has been mapped to residue 8-12 (Riek, R. et al. Eur. J. Biochem. 2001, 268, 5930-5936). Despite their different aggregation behavior monomeric Aβ1-40 and Aβ1-42 have very similar secondary structures with the exception that the longer variant is more rigid in its C-terminal (Yan, Y.; Wang, C. J. Mol. Biol. 2006, 364, 853-862). In SDS-micelles, Aβ1-40 has been shown to form two helices, covering residues 15-24 and 30-35 respectively (Jarvet, J. et al. J. Biomol. NMR 2007, 39, 63-72). In the micelles, the first helix is superficially located and the second helix is buried in the hydrophobic interior. Helix formation, in similar locations of the peptide, has also been observed in both Aβ1-40 and Aβ1-42 using structure-inducing solvents such as trifluorethanol and hexafluoro-isopropanol (Crescenzi, O. et al. Eur J Biochem 2002, 269, 5642-5648) and at physiological salt concentrations (Subramanian Vivekanandan et al. Biochemical and Biophysical Research Communications 2011, 411, 312-316).
Finding ways of inhibiting Aβ misfolding and amyloid formation is important but challenging and several strategies have been proposed. The aggregation process of Aβ is not fully understood and it is unclear which forms of Aβ that are toxic. Earlier it was believed that the mature fibrils were the main cause of the disease (Hardy, J. A.; Higgins, G. A. Science 1992, 256, 184-185) and a number of inhibitors of fibril formation have been reported. However, more and more findings point to the toxic nature of soluble oligomers produced early in the aggregation pathway (Hardy, J.; Selkoe, D. J. Science 2002, 297, 353-356). These early aggregates are not structurally defined making inhibitor design a difficult task and it is also possible that targeting species on the fibrillation pathway may result in accumulation of toxic oligomers. A more appealing idea would be to target and stabilize an Aβ-monomer, thereby preventing misfolding and subsequent amyloid formation.
Aβ contains a discordant helix (residue 16-23) i.e. a helix composed of amino acids with a high propensity for β-strand conformation (Kallberg, Y. et al. J. Biol. Chem. 2001, 276, 12945-12950). Peptides derived from this region form fibrils and, in Aβ, this region has been found essential for fibril formation (Liu, R. et al. J. Neurosci. Res. 2004, 75, 162-171). It has previously been shown that by using small designed ligands, directed towards the discordant region of Aβ (residues 13-23), it is possible to stabilize a helical structure and reduce aggregation in vitro (Nerelius, C. et al. Proc. Natl. Acad. Sci. U.S.A 2009, 106, 9191-9196). These ligands also reduced cell toxicity of Aβ and prevented Aβ-induced reduction of γ oscillations of hippocampal slices. Oral administration of two of these compounds in a Drosophila model of Alzheimer's disease (Crowther, D. C. et al. Neuroscience 2005, 132, 123-135) increases longevity, decreases locomotor dysfunction and reduces neuronal damage (Nerelius, C. et al. Proc. Natl. Acad. Sci. U.S.A 2009, 106, 9191-9196). These results indicate that this approach holds promise for the development of orally available compounds against Alzheimer's disease. Additional support for the concept comes from recent molecular dynamics simulations that also uncover details of the mechanism of unfolding of the Aβ central helix (Ito, M. et al. PLoS One 2011, 6, e17587) as well as retardation of the folding in presence of ligands designed to interact with the native helical conformation (Ito, M. et al. PLoS One 2012, 7, e30510).
The inventors have developed a number of new ligands designed to have more extended interaction with Aβ, through interaction with several both hydrophobic and polar regions across the central part of the peptide. In particular, the new ligands are designed to have higher affinity to helical Aβ in order to reduce the Aβ associated neurotoxicity. The synthesis strategy also involves a number of novel amino acids which allows for substantial variation of substituents and hence makes it possible to fine-tune the structures further.